R&D Systems Tools for Cell Biology Research™

Products for Research The Chemokine Superfamily are small cell surface-localized or secreted chemotactic that bind to and activate specifi c G -coupled chemokine receptors. Most chemokines have at least four conserved N-terminal cysteine residues that form two intramolecular disulfi de bonds. Four chemokine subfamilies (CXC, CC, C and CX3C) have been defi ned based upon the placement of the fi rst two cysteine residues. The CXC chemokine subfamily is characterized by two cysteine residues separated by one amino acid. Within this subfamily, two CXC classes are further defi ned by the presence or absence of an ELR motif sequence. ELR– CXC chemokines act as chemoattractants for lymphocytes, while ELR+ CXC chemokines are chemoattractants for . Additionally, CXC chemokines can mediate angiogenesis.1 The CC chemokine subfamily is defi ned by two adjacent cysteine CCR1 H: CCL3-5, 7, 8, 13-16, 23, CCL3L1, CCL3L3, CCL4L1, CCL4L2 M: CCL3-7, 9/10 residues. CC chemokines induce infl ammatory H: XCL1, 2 XCR1 responses via regulation of monocyte, M: XCL1 macrophage, mast cell, and T cell migration.2 H: CCL2, 7, 8, 13, 16 CCR2 (A or B) M: CCL2, 7, 12 C chemokines are characterized by a single H: CCL26 CX3CR1 cysteine residue and are constitutively expressed H: CX3CL1 in the thymus where they regulate T cell H: CCL5, 7, 8, 11, 13, 14, 15, 24, 26, 28, CCL3L1, CCL3L3 M: CX3CL1 CCR3 M: CCL5, 7, 9/10, 11, 24 diff erentiation.3 The CX3C chemokine subfamily is defi ned by two cysteine residues separated by H: CXCL6-8 CXCR1 three amino acids. Cell surface-localized CX3CL1/ H: CCL2, 3, 5, 17, 22 M: CXCL7, LIX CCR4 Fractalkine mediates leukocyte adhesion while M: CCL2, 3, 5, 17, 22 soluble CX3CL1/Fractalkine is chemotactic for H: CXCL1-3, 5-8 leukocytes.4 CX3CL1/Fractalkine is also a critical H: CCL3-5, 8, 11, 13, 14, 16, CCL3L1, M: CXCL1-3, 7, LIX CXCR2 CCR5 CCL3L3, CCL4L1, CCL4L2 regulator of microglia-neuron communication M: CCL3-5 during neural development.5 H: CXCL4, 9-11, 13, CXCL4L1 While chemokine receptors generally bind only CCR6 H: CCL20 M: CXCL4, 9-11 CXCR3 M: CCL20 one subfamily of chemokines, within those subfamilies, most chemokines display promiscuous receptor binding patterns. The CCR7 H: CCL19, 21 M: CCL19, 21 H: CXCL4, 9-11, CXCL4L1 CXCR3B redundancy of chemokine ligand-receptor binding may ensure robust signaling. In addition, promiscuous binding and non-signaling CCR8 H: CCL1, 4, 16, 17 M: CCL1, 8 H: CXCL12 CXCR4 chemokine receptors off er mechanisms by which M: CXCL12 chemokine signaling can be regulated by either subtle diff erences in receptor signaling or H: CCL25 CCR9 M: CCL25 diff erences in ligand-receptor expression H: CXCL13 M: CXCL13 CXCR5 patterns.6 Select chemokine ligands and receptors are implicated in HIV infection and H: CCL27, 28 CCR10 M: CCL27, 28 persistence, while aberrant chemokine H: CXCL16 expression and signaling is associated with M: CXCL16 CXCR6 pathological conditions including infl ammatory CCR11 H: CCL2, 8, 13, 19, 21, 25 diseases and cancer.7-9 M: CCL19, 21, 25 References H: CXCL11, 12 CXCR7 1. Mehrad, B. et al. (2007) Thromb. Haemost. 97:755. M: CXCL11, 12 PITPNM3 H: CCL18 2. Ono, S.J. et al. (2003) J. Allergy Clin. Immunol. 111:1185. 3. Lei, Y. & Y. Takahama (2012) Microbes Infect. 14:262. H: CXCL14, 17 4. Jones, B.A. et al. (2010) Mol. Interv. 10:263. M: CXCL14, 17 Unknown 5. Wolf, Y. et al. (2013) Front. Cell. Neurosci. :26. 7 CCRL2/CRAM-A/B H: CCL19 M: CXCL15 6. Bennett, L.D. et al. (2011) Immunology 134:246. 7. Evans, V.A. et al. (2012) Rev. 23:151. 8. Balkwill, F.R. (2012) J. Pathol. 226:148. H: CCL1, 2, 5, 7, 8, 11, 13, 14, 16-18 9. White, G.E. et al. (2013) Pharmacol. Rev. 65:47. CCRL2/LCCR M: CCL19 M: CCL2, 11 H: CXCL1-3, 5, 6, 8 DARC M: CXCL1, 2, LIX H: CXCL4, 9-11, 13 D6 H: CCL2-5, 7, 8, 11, 13, 14, 17, 22, CCL3L1 M: CCL2, 3

Key H: Human M: Mouse C Chemokines/Receptors CXC ELR+ Chemokines/Receptors For additional information CC Chemokines/Receptors CX3C Chemokines/Receptors >RnDSystems.com/ChemokineInfoTable CXC ELR- Chemokines/Receptors Non-signaling Chemokine Receptors

For research use only. Not for use in diagnostic procedures. CXC Chemokines CXC Chemokines Recombinant & Recombinant CXC chemokines, also known as  chemokines, Molecule Natural Antibodies ELISAs Human Protein Sampler Packs are critical regulators of normal development CXCL1/2/3/GRO CR H (B/N, FC, WB) CR (B/N, WB) and immune responses. The CXC chemokine subfamily consists of nineteen ligands and H (B/N, E, FC, WB) M (B/N, E, WB) CXCL1/GRO/KC/CINC-1 H M R H M R R (B/N, IHC, WB) seven receptors. CXC chemokines that contain an ELR motif are potent angiogenic H (B/N, FC, WB) M (B/N, E, ICC, IHC, WB) CXCL2/GRO/MIP-2/CINC-3 H M R CR M R factors, while ELR–CXC chemokines are R (B/N, E, WB) angiostatic. Select CXC chemokines and CXCL3/GRO/CINC-2/DCIP-1 H M R H (B/N, FC, WB) M (B/N, WB) R (B/N, E, WB) R receptors regulate hematopoietic stem cell CXCL4/PF4 H M H (E, FC, WB) M (E, WB) H M migration. They are also involved in regulating CXCL5/ENA-70 H tumor progression and are of interest as targets 1-3 CXCL5/ENA-74 H for therapeutic intervention. References CXCL5/ENA-78 HH (B/N, E, FC, IHC, WB) H 1. Zhu, Q. et al. (2012) J. Mol. Histol. 43:699. CXCL6/GCP-2 HH (B/N, E, FC, WB) H 2. Vinader, V. & K. Afarinkia (2012) Future Med. Chem. 4:853. CXCL7/NAP-2 HH (B/N, E, WB) H 3. Owen, J.L. & M. Mohamadzadeh (2013) Front. Physiol. 4:159. CXCL7/Thymus Chemokine-1 M R M (B/N, E, IHC, WB) R (B/N, E, IHC, WB) M R 104 H (B/N, E, FC, ICC, IHC, WB) P (B/N, E, WB) CXCL8/IL-8 H P Ca F H P Ca F Ca (B/N, E, ICC, WB) F (B/N, E, ICC, WB) 103 CXCL9/MIG H M H (B/N, E, FC, ICC, WB) M (B/N, E, WB) H M H (B/N, E, FC, ICC, WB) M (B/N, E, IHC, WB) CXCL10/IP-10/CRG-2 H M CR H M  CR (B/N, WB) 102 CXCL11/I-TAC H M H (B/N, E, WB) M (B/N, E, WB) H M  CXCR5 CXCL12/SDF-1 H M F RM H (B/N, E, FC, IHC, WB) M (B/N, E, FC, IHC, WB) H M  1 CXCL12/SDF-1 H M F RM H M 10 CXCL12/SDF-1 H F H (B/N, E, WB)  CXCL12/SDF-1 H 100 CXCL13/BLC/BCA-1 H M H (B/N, E, FC, IHC, WB) M (B/N, E, IHC, WB) H M 100 101 102 103 104 B220/CD45R CXCL14/BRAK H M H (E, WB) M (WB) H Detection of CXCR5 and B220/CD45R on Mouse Splenocytes by Flow CXCL15/Lungkine MM (E, WB) M Cytometry. Mouse splenocytes were stained for CXCR5 and B220/CD45R expression using a PE-conjugated Rat Anti-Mouse CXCR5 Monoclonal Anti- CXCL16 H M H (B/N, E, FC, IHC, WB) M (B/N, E, FC, WB) H M body (Catalog # FAB6198P) and an APC-conjugated Rat Anti-Mouse B220/ CXCL17/VCC-1 H M H (FC, ICC, WB) M (WB)  CD45R Monoclonal Antibody (Catalog # FAB1217A). Quadrants were set based on isotype controls. LIX M R M (B/N, E, WB) R (B/N, E, WB) M R CXCR1/IL-8 RA Antibody (μg/mL) 10-2 10-1 100 101 CXC Chemokine Receptors 6000 6000 Molecule Antibodies Tocris Biochemicals & Peptides 5000 5000 CXCR1/IL-8 RA H (B/N, FC, IHC) Cell Migration (Mean RFU) CXCR2/IL-8 RB H (B/N, FC, IHC) M (B/N, FC)  4000 4000 CXCR3 H (B/N, FC, IHC) M (FC) 3000 3000 CXCR4 H (B/N, FC, IHC) M (B/N, FC, ICC, IHC) F (FC, ICC)  CXCR5 H (B/N, FC, ICC, IHC) M (FC, ICC) 2000 2000 Cell Migration (Mean RFU) Migration Cell CXCR6 H (FC) M (FC) 1000 1000 CXCR7/RDC-1 H (FC, IHC) M (FC, WB)  DARC H (FC, ICC) M (FC, WB) R (FC, WB) 0 0 10-2 10-1 100 101 CXCL8/IL-8 (ng/mL) Chemotaxis Induced by CXCL8/IL-8 and Neutralization using a Human CXCR1/IL-8 RA Antibody. Recombinant Human CXCL8/IL-8 (Catalog # 208-IL) chemoattracts the BaF3 mouse pro-B cell line transfected with hu- man CXCR1/IL-8 RA in a dose-dependent manner (orange line). The amount of cells that migrated through to the lower chemotaxis chamber was mea- sured by Resazurin (Catalog # AR002). Chemotaxis elicited by 1 ng/mL Species Key: H Human, M Mouse, R Rat, B Bovine, Ca Canine, CR Cotton Rat, F Feline, P Porcine, RM Rhesus Macaque, V Viral Recombinant Human CXCL8/IL-8 was neutralized by increasing concentra- Application Key: B/N Blocking/Neutralization, E ELISA (Cap. or Det.), FC Flow Cytometry, ICC Immunocytochemistry, IHC Immunohistochemistry, IP Immunoprecipitation, tions of Human CXCR1/IL-8 RA Monoclonal Antibody (Catalog # MAB330; WB Western Blot green line).

RnDSystems.com/Chemokines CC Chemokines CC Chemokines Recombinant CC chemokines, also known as  chemokines, Molecule Recombinant & Antibodies ELISAs Human Protein are critical mediators of the infl ammatory Natural Proteins Sampler Packs response. The CC subfamily contains thirty-four CCL1/I-309/TCA-3 H M H (B/N, E, WB) M (B/N, E, WB) H M  ligands and eleven receptors. Many of these are H (B/N, E, FC, IHC, WB) M (B/N, E, FC, WB) CCL2/JE/MCP-1 H M R Ca H M R Ca  implicated in chronic infl ammatory diseases Ca (B/N, E, ICC, WB) CR (WB) including rheumatoid arthritis, atherosclerosis, H (B/N, E, FC, ICC, IHC, WB) M (B/N, E, FC, ICC, and metabolic syndrome.1 CC chemokines also CCL3/MIP-1 H M R CR IHC, WB) R (B/N, IHC, WB) Ca (WB) H M Ca  regulate the recruitment of leukocytes to the CR (B/N, WB) tumor microenvironment, particularly tumor- associated macrophages and myeloid-derived CCL3L1/MIP-1 Isoform LD78 H 2 H (B/N, E, FC, ICC, IHC, WB) M (B/N, IHC, WB) suppressor cells. CCL4/MIP-1 H M R Ca CR H M  R (B/N, WB) Ca (ICC, WB) CR (B/N, WB) References 1. White, G.E. et al. (2013) Pharmacol. Rev. 65:47. CCL4L1/LAG-1 H 2. Mantovani, A. et al. (2010) Cytokine Growth Factor Rev. H (B/N, E, FC, ICC, IHC, WB) M (B/N, E, ICC, WB) CCL5/RANTES H M Ca CR F H M R Ca F  21:27. Ca (B/N, ICC) CR (B/N, WB) F (B/N, E, WB) CCL20/MIP-3D Antibody (μg/mL) 10-1 100 101 CCL6/C10 MM (B/N, E, FC, WB) M

6000 6000 CCL7/MCP-3/MARC H M H (B/N, E, FC, WB) M (B/N, WB) H CCL8/MCP-2 HH (B/N, E, WB) M (WB) H M 5000 5000 Cell Migration (Mean RFU) CCL9/10/MIP-1 MM (B/N, E, WB) M 4000 4000 CCL11/Eotaxin H M H (B/N, E, FC, IHC, WB) M (B/N, E, IHC, WB) H M  CCL12/MCP-5 MM (B/N, E, WB) M 3000 3000 CCL13/MCP-4 HH (B/N, E, FC, WB) H 2000 2000

Cell Migration (Mean RFU) Migration Cell CCL14/HCC-1/HCC-3 H (B/N, E, WB)

1000 1000 CCL14a/HCC-1 HH (B/N, E, WB) H CCL14b/HCC-3 H (B/N, E, WB) 0 0 10-1 100 101 102 CCL15/MIP-1 HH (B/N, E, WB) H CCL20/MIP-3 (ng/mL) D CCL16/HCC-4 HH (B/N, E, FC, IHC, WB) H Chemotaxis Induced by CCL20/MIP-3 and Neutralization using a Hu- man CCL20/MIP-3 Antibody. Recombinant Human CCL20/MIP-3 (Cat- CCL17/TARC H M H (B/N, E, FC, IHC, WB) M (B/N, E, WB) H M  alog # 360-MP) chemoattracts the BaF3 mouse pro-B cell line transfected CCL18/PARC HH (E, FC, WB) H with human CCR6 in a dose-dependent manner (orange line). The amount of cells that migrated through to the lower chemotaxis chamber was mea- CCL19/MIP-3 H M R H (B/N, E, FC, ICC, IHC, WB) M (B/N, E, ICC, WB) H M  sured by Resazurin (Catalog # AR002). Chemotaxis elicited by 10 ng/mL H (B/N, E, FC, ICC, IHC, WB) M (B/N, E, FC, WB) Recombinant Human CCL20/MIP-3 is neutralized by increasing CCL20/MIP-3 H M R H M R  concentrations of Human CCL20/MIP-3 Monoclonal Antibody R (B/N, E, WB) (Catalog # MAB360; green line). CCL21/6Ckine H M R H (B/N, E, ICC, WB) M (B/N, E, FC, IHC, WB) H M  CCL22/MDC H M H (B/N, E, FC, WB) M (B/N, E, WB) H M  CCL23/Ck8-1 HH (B/N, E, WB) H CCL23/MPIF-1 HH (B/N, E, FC, WB) H  CCL24/Eotaxin-2/MPIF-2 H M H (B/N, E, IHC, WB) M (B/N, E, WB) H M  CCL25/TECK H M H (B/N, E, WB) M (B/N, E, IHC, WB) H M CCL26/Eotaxin-3 HH (B/N, E, IHC, WB) H  CCL26-like/Eotaxin-3-like R (WB) CCL27/CTACK H M R H (B/N, E, IHC, WB) M (B/N, E, IHC, WB) H M CCL28 H M H (B/N, E, WB) M (B/N, E, WB) H M

Detection of CCL3/MIP-1 in Rat Kidney. Perfusion-fi xed frozen sections of rat kidney were stained for CCL3/MIP-1 expression using a Mouse Anti- Rat CCL3/MIP-1 Monoclonal Antibody (Catalog # MAB66251). The tissue was stained using the NorthernLights™ 557-conjugated Donkey Anti- Mouse IgG Secondary Antibody (Catalog # NL007; red) and counterstained Species Key: H Human, M Mouse, R Rat, B Bovine, Ca Canine, CR Cotton Rat, F Feline, P Porcine, RM Rhesus Macaque, V Viral with DAPI (blue). Specifi c staining was localized to the plasma membranes Application Key: B/N Blocking/Neutralization, E ELISA (Cap. or Det.), FC Flow Cytometry, ICC Immunocytochemistry, IHC Immunohistochemistry, IP Immunoprecipitation, of epithelial cells in convoluted tubules. WB Western Blot For research use only. Not for use in diagnostic procedures. CC Chemokine Receptors C Chemokines Molecule Antibodies Tocris Biochemicals & Peptides C chemokines, also known as  chemokines, CCR1 H (FC, IHC) M (FC, WB)  mediate homing and leukocyte maturation CCR2 H (FC) M (FC)  within lymphoid tissues. This subfamily CCR3 H (B/N, FC, IHC) M (FC)  consists of two ligands, XCL1 and XCL2, CCR4 H (FC, WB) M (WB) R (FC)  which both bind to XCR1. The XCR1 receptor is expressed on a unique subset of dendritic CCR5 H (B/N, FC, ICC, IHC, IP, WB) M (FC, WB) R (FC)  cells (DCs) that cross-presents antigens to CCR6 H (FC, IHC) M (FC, IHC) T cells.1-3 The specifi city of XCR1 expression on CCR7 H (B/N, FC, ICC) M (B/N, FC, ICC) cross-presenting DCs makes it an attractive CCR8 H (B/N, FC) M (WB) R (FC, WB)  target for antigen delivery by DC-targeted 1,4 CCR9 H (FC, ICC, IHC) M (FC) R (FC) cancer vaccines. CCR10 H (FC) M (FC) R (FC) References 1. Kroczek, R.A. & V. Henn (2012) Front. Immunol. 3:14. CCR11 H (FC) 2. Yamazaki, C. et al. (2013) J. Immunol. 190:6071. CCRL2/CRAM-A/B H (FC) 3. Bachem, A. et al. (2012) Front. Immunol. 3:214. 4. Haniff a, M. et al. (2013) Oncoimmunology 2:e23140. CCRL2/LCCR M (FC, ICC) D6 H (FC) CC Chemokine-related Molecules CX3C Chemokines Molecule Recombinant & Natural Proteins Antibodies The CX3C chemokine subfamily, also known as the  subfamily, regulates leukocyte CCI VV (B/N, WB) homing and adhesion. There is only one CX3C MCK-2 V (WB) chemokine ligand, CX3CL1/Fractalkine, which MCV-type II Chemokine-like VV (B/N, WB) binds to CX3CR1. CX3CL1/Fractalkine mediates Protein an infl ammatory response and appears to MIP-I VV (B/N, WB) play a signifi cant role in infl ammatory dis- MIP-II VV (WB) orders including atherosclerosis, allergen- induced asthma, and neuro-infl ammation.1-4 MIP-III V (WB) References TAFA1/FAM19A1 HH (B/N, IHC, WB) 1. Bordon, Y. (2010) Nat. Rev. Immunol. 10:810. TAFA2/FAM19A2 HH (B/N, IHC, WB) 2. Flierl, U. & A. Schafer (2012) Thromb. Haemost. 108:457. TAFA3/FAM19A3 H (FC, WB) 3. Julia, V. (2012) Allergy 67:1106. 4. Kiguchi, N. et al. (2012) Curr. Opin. Pharmacol. 12:55. TAFA4/FAM19A4 HH (FC, WB) 104 TAFA5/FAM19A5 HH (FC, IHC, WB) M (FC, WB) R (FC, WB) C Chemokine & Receptor

Molecule Recombinant & Natural Proteins Antibodies ELISAs 103 XCL1/Lymphotactin H M H (B/N, E, WB) M (B/N, E, IHC, WB) H M XCR1 H (FC, ICC, WB) CX3C Chemokine & Receptor 102 CX3CR1 Molecule Recombinant & Natural Proteins Antibodies ELISAs H (B/N, E, FC, IHC, WB) M (B/N, E, WB) CX3CL1/Fractalkine H M R H M R 101 R (B/N, E, IHC, WB) CX3CR1 H (FC, WB) M (FC, WB)

Other Chemotactic Molecules 100 100 101 102 103 104 Molecule Recombinant & Natural Proteins Antibodies ELISAs Integrin DX/CD11c Detection of CX3CR1 and Integrin X/CD11c on Mouse Splenocytes by H M H (E, FC, WB) M (B/N, E, FC, WB) H M Flow Cytometry. Mouse splenocytes were stained with an APC-conjugated ChemR23 H (FC) M (FC) Goat Anti-Mouse CX3CR1 Antigen Affi nity-purifi ed Polyclonal Antibody (Catalog # FAB5825A) and a PE-conjugated anti-mouse Integrin X/ CD11c antibody.

RnDSystems.com/Chemokines Multianalyte Profi ling Kits for Chemokine Research

R&D Systems off ers both membrane-based and bead-based multianalyte profi ling kits for chemokine research. These kits off er highly effi cient, economical assays for simultaneously detecting multiple chemokines in a single sample. Proteome Profi ler™ Antibody Arrays The Proteome Profi ler™ Human and Mouse Chemokine Antibody Arrays are nitrocellulose membrane-based arrays that allow for the simultaneous detection of 31 human or 25 mouse chemokines in a single sample. Proteome Profi ler Antibody Arrays eliminate the need for multiple immunoprecipitation/Western blot experiments and require no specialized equipment. For additional information, please visit RnDSystems.com/ProteomeProfi ler.

Proteome Profi ler Antibody Arrays Human Chemokine Antibody Array (Catalog # ARY017) Contains 4 membranes  each spotted in duplicate with 31 diff erent chemokine antibodies CCL1/I-309, CCL2/MCP-1, CCL3/CCL4 (MIP-1/MIP-1), CCL5/RANTES, CCL7/MCP-3, CCL14/HCC-1/HCC-3, CCL15/MIP-1/LKN-1, CCL17/TARC, CCL18/ PARC, CCL19/MIP-3, CCL20/MIP-3, CCL21/6Ckine, CCL22/MDC, CCL26/Eotaxin-3, CCL28, CX3CL1/Fractalkine, CXCL1/GRO, CXCL4/PF4, CXCL5/ Light ENA-78, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, CXCL16, CXCL17/VCC-1, XCL1/Lymphotactin, Chemerin, Detection Antibody IL-16, Midkine Streptavidin-HRP, Protein Chemiluminescence Mouse Chemokine Antibody Array (Catalog # ARY020) Contains 4 membranes  each spotted in duplicate with 25 diff erent chemokine antibodies CCL2/JE/MCP-1, CCL3/CCL4 (MIP-1/MIP-1), CCL5/RANTES, CCL6/C10, CCL8/MCP-2, CCL9/10/MIP-1, CCL11/Eotaxin, CCL12/MCP-5, CCL21/6Ckine, CCL22/MDC, CCL27/CTACK, CCL28, CX3CL1/Fractalkine, CXCL1/KC, CXCL2/MIP-2, CXCL9/MIG, CXCL10/IP-10/CRG-2, CXCL11/I-TAC, CXCL12/SDF-1, Capture Antibody CXCL13/BLC/BCA-1, CXCL16, Chemerin, Complement Component C5/C5a, IL-16, LIX Array Membrane Luminex Screening and Performance Assays Luminex Screening and Performance Assays are bead-based multianalyte profi ling kits. These kits utilize color-coded polystyrene or superparamagnetic microparticles that are coated with analyte-specifi c antibodies. Captured analytes are subsequently detected using a cocktail of biotinylated detection antibodies and a streptavidin-phycoerythrin conjugate. Luminex Screening Assays are our most fl exible kits for multianalyte profi ling of up to 100 human or 42 mouse analytes. Luminex Performance Assays are our most accurate and precise bead-based multianalyte profi ling kits. These assays are optimized for select panels of analytes. For additional information, please visit RnDSystems.com/Luminex.

Chemokine Luminex Screening and Performance Assay Analytes Human Luminex Screening Assay Analytes CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1, CCL5/RANTES, CCL8/MCP-2, CCL13/MCP-4, CCL17/TARC, CCL20/MIP-3, CXCL1/GRO, CXCL4/PF4, CXCL5/ENA-78, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CXCL13/BLC/BCA-1 Analyte Mouse Luminex Screening Assay Analytes CCL2/JE, CCL3/MIP-1, CCL4/MIP-1, CCL5/RANTES, CCL20/MIP-3, CXCL1/KC, CXCL2/MIP-2, CXCL10/IP-10/CRG-2, CXCL12/SDF-1, LIX Human Cytokine Panel A Luminex Performance Assay Analytes*

CCL2/MCP-1, CCL3/MIP-1, CCL4/MIP-1, CCL5/RANTES, CXCL5/ENA-78, CXCL8/IL-8, FGF basic, G-CSF, GM-CSF, IFN-, IL-1/IL-1F1, IL-1/IL-1F2, Streptavidin-PE IL-1ra/IL-1F3, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, TNF-, Thrombopoietin/Tpo, VEGF Biotin Detection Antibody Mouse Cytokine Panel Luminex Performance Assay Analytes Target Analyte Capture Antibody CCL2/JE/MCP-1, CXCL1/KC, CXCL2/MIP-2, GM-CSF, IFN-, IL-1/IL-1F2, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, p70, IL-13, IL-17, TNF-, VEGF Rat Cytokine Panel Luminex Performance Assay Analytes Luminex Bead CXCL2/CINC-3, CXCL3/CINC-2/, GM-CSF, ICAM-1/CD54, IFN-, IL-1/IL-1F1, IL-1/IL-1F2, IL-2, IL-4, IL-6, IL-10, IL-13, IL-18/IL-1F4, L-Selectin/ CD62L, TIMP-1, TNF-, VEGF * Analytes in this panel are available in both the polystyrene and magnetic bead formats.

4500 Untreated 4000 PMA 3500 3000 2500 Detection of Multiple Chemokines in Cell Culture Supernates Using the Human Luminex Screening Assay. The human THP-1 monocytic leukemia 2000 cell line was treated with phorbol 12-myristate 13-acetate (PMA) for 24 1500 hours. Cell culture supernates were collected from untreated and treated

Concentration (pg/mL) Concentration cells and the levels of multiple chemokines were simultaneously deter- 1000 mined using the Human Luminex Screening Assay (Catalog # LXSAH). Of the 500 16 chemokines tested, 9 gave measurable values in each of the PMA-stimu- N.D. N.D. N.D. N.D. lated samples. The average values and standard deviations for the detect- 0 able analytes are shown. N.D. Not Detected. (Figure on left). CCL2/MCP-1 CCL3/MIP-1D CCL4/MIP-1E CCL5/RANTES CCL20/MIP-3D CXCL1/GROD CXCL5/ENA-78 CXCL8/IL-8 CXCL10/IP-10

For research use only. Not for use in diagnostic procedures. Fluorokine® Receptor Detection Kits for Chemokine Research

Fluorokine Receptor Detection Kits utilize recombinant biotinylated ligands to identify cytokine receptors by fl ow cytometry. These kits can be used to determine the percentage of cells expressing 150 a specifi c target receptor within a population of cells or to estimate the density of the target receptor on the cell surface. For additional information, please visit RnDSystems.com/FluoroReceptorKit.

Kit Species Catalog # Features 100 CCL1/I-309 Human NFCC10  Biotinylated ligands mirror physiological ligand/ CCL2/MCP-1/JE Human NFCP0 receptor interactions

Mouse NFJE0  Determine the percentage of cells expressing target Number Cell Relative 50 CCL3/MIP-1 Human NFLD0 receptors within a population Mouse NFM1A0  Estimate the target receptor density on cell surfaces CCL4/MIP-1 Human NFBM0  Optimized for use with both cultured cells and 0 CCL5/RANTES Human NFRN0 peripheral blood cells 102 103 104 105 CCL21/6Ckine CCL11/Eotaxin Human NFEO0 Detection of CCL21/6Ckine Expression on Human CD4+ T cells. Human CCL17/TARC Human NFTC0 CD4+ T cells were isolated from peripheral blood mononuclear cells using + Mouse NFMTC0 the MagCellect Human CD4 T Cell Isolation Kit (Catalog # MAGH102). Within the isolated CD4+ T cell population, the Human CCL21/6Ckine Bioti- CCL19/MIP-3 Human NFCCL19 nylated Fluorokine Kit (Catalog # NFCCL21) was used to detect CCL21/6Ckine receptor positive cells. The cells were stained with biotinylated recombinant CCL21/6Ckine Human NFCCL21 Fluorescein-conjugated Avidin human CCL21/6Ckine (light green histogram) or with the biotinylated CCL25/TECK Human NFTK0 Biotinylated Ligand negative control protein (open histogram). The specifi city of the assay was A demonstrated by staining the cells in the presence of the anti-human Receptor of Interest FITC CX3CL1/Fractalkine Human NFCX310 B CCL21/6Ckine blocking antibody provided in the kit (dark green histogram). CXCL8/IL-8 Human NF800 CXCL12/SDF-1 Human NNS00

Biochemicals and Peptides for Chemokine Research

Chemokine receptor activity can be studied in vivo and in vitro using biochemical and peptide agonists and antagonists which modulate their activity. These G protein-coupled receptors (GPCRs) are highly targetable, and a variety of potent and selective agonists and antagonists have been developed to facilitate chemokine research. products available from Tocris Bioscience are listed below.

Chemokine Receptor Agonists Chemokine Receptor Antagonists AMD 3100 (μM) 10-4 10-3 10-2 10-1 100 101 102 Receptor Product Name Catalog # Receptor Product Name Catalog # 6000 6000 CCR8 ZK 756326 2565 CCR3 SB 297006 4213

5000 Cell Migration (Mean RFU) CXCR7 TC 14012 4300 SB 328437 3650 5000

UCB 35625 2757 4000 4000 Chemokine Receptor Antagonists CCR4 C 021 dihydrochloride 3581 3000 Receptor Product Name Catalog # CCR5 DAPTA 2423 3000 CCR1 BX 471 3496 3756

Cell Migration (Mean RFU) Migration Cell 2000 2000 BX 513 hydrochloride 2769 CXCR2 SB 225002 2725 J 113863 2595 SB 265610 2724 1000 1000 CXCR4 AMD 3100 octahydrochloride 3299 UCB 35625 2757 0 0 CCR2 BMS CCR2 22 3129 AMD 3465 hexahydrobromide 4179 10-4 10-3 10-2 10-1 100 101 102 CXCL12/SDF-1D (ng/mL) INCB 3284 dimesylate 4306 FC 131 4320 CXCL12/SDF-1-induced Chemotaxis Is Antagonized by AMD 3100 RS 504393 2517 IT1t dihydrochloride 4596 Octahydrochloride. Increasing concentrations of Recombinant Human/ Feline/Rhesus Macaque CXCL12/SDF-1 (Catalog # 350-NS) were used to CCR2B RS 102895 hydrochloride 2089 TC 14012 4300 stimulate chemotaxis of the BaF3 mouse pro-B cell line transfected with Teijin compound 1 3664 WZ 811 3951 human CXCR4. Cells that migrated to the lower compartment of a chemo- taxis chamber were measured using the redox sensitive dye, Resazurin (Catalog # AR002; red line). The eff ect induced by 1 ng/mL Recombinant Human/Feline/Rhesus Macaque CXCL12/SDF-1 was antagonized with increasing concentrations of the highly selective CXCR4 inhibitor, AMD 3100 octahydrochloride (Catalog # 3299; blue line).

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Peer-Reviewed References Using R&D Systems Chemokine-Related Products 1. Igoucheva, O. et al. (2013) Immunotargeting and eradication 3. Rath, T. et al. (2012) Serum proteome profi ling identifi es 5. Harris, T.H. et al. (2012) Generalized Lévy walks and the role of melanoma using a chemokine-enhanced DNA vaccine. novel and powerful markers of cystic fi brosis liver disease. of chemokines in migration of eff ector CD8+ T cells. Nature Ther. [Epub ahead of print]. PLoS One 8:e58955. 486:545. Recombinant Mouse CCL20/MIP-3 (Catalog # 760-M3) Proteome Profi ler Human Chemokine Array Kit APC-conjugated Mouse Anti-Human CXCR3 Recombinant Mouse CCL21/6Ckine (Catalog # 457-6C) (Catalog # ARY017) Monoclonal Antibody (Catalog # FAB160A) Proteome Profi ler Human Cytokine Array Kit, Panel A Sample: Mouse brain mononuclear cells Sample: Mouse (Catalog # ARY005) Application: In vivo Application: Flow cytometry Proteome Profi ler Human sReceptor Array Kit, 2. Soehnlein, O. et al. (2013) Distinct functions of chemokine Non- Hematopoietic Panel (Catalog # ARY012) Recombinant Mouse CXCL9/MIG (Catalog # 492-MM) receptor axes in the atherogenic mobilization and Recombinant Mouse CXCL10/IP-10/CRG-2 Sample: Human serum recruitment of classical monocytes. EMBO Mol. Med. 5:471. (Catalog # 466-CR) Application: Multianalyte profi ling Recombinant Mouse CXCL11/I-TAC (Catalog # 572-MC) Rat Anti-Mouse CXCL1/KC Monoclonal Antibody Human TIMP-4 Quantikine ELISA Kit (Catalog # DTM400) (Catalog # MAB4531) Sample: Mouse splenocytes and brain mononuclear cells Human Endoglin/CD105 Quantikine ELISA Kit Rat IgG Isotype Control (Catalog # MAB006) Assay: Cell migration 2A (Catalog # DNDG00) Sample: Mouse Human HGF Quantikine ELISA Kit (Catalog # DHG00) 6. Holub, M. et al. (2013) Cytokines and chemokines as Application: In vivo biomarkers of community-acquired bacterial infection. Sample: Human serum Mediators Infl amm. 2013:190145. Rat Anti-Mouse CXCR2 Monoclonal Antibody Application: ELISA (Catalog # MAB2164) Human Cytokine Premixed Luminex Performance 4. Patel, K. et al. (2012) The GHS-R blocker D-[Lys3] GHRP-6 Goat Anti-Human/Mouse CX3CR1 Antigen Affi nity- Assay Kit A (Catalog # FCST03) serves as CCR5 chemokine receptor antagonist. Int. J. Med. purifi ed Polyclonal Antibody (Catalog # AF5825) Sci. 9:51. Sample: Human serum Sample: Mouse blood-, bone marrow-, spleen-, and Application: Multianalyte quantifi cation Human CCL3/MIP-1 Biotinylated Fluorokine Kit aorta-derived mononuclear cells  (Catalog # NFLD0) 7. Miller, R.E. et al. (2012) CCR2 chemokine receptor signaling Application: Flow cytometry Human CCL4/MIP-1 Biotinylated Fluorokine Kit mediates pain in experimental osteoarthritis. Proc. Natl. Human CXCL1/GRO Quantikine® ELISA Kit (Catalog # NFBM0) Acad. Sci. USA 109:20602. (Catalog # DGR00) Human CCL5/RANTES Biotinylated Fluorokine Kit Tocris CCR2 Inhibitor RS 504393 (Catalog # 2517) (Catalog # NFRN0) Sample: Human plasma Tocris COX-1 Inhibitor Indomethacin (Catalog # 1708) Application: ELISA Sample: Human T-lymphoblastoid cells (CEM.NKR) Sample: Mouse Application: Receptor binding assay Mouse/Rat CCL2/JE/MCP-1 Quantikine ELISA Kit Application: In vivo (Catalog # MJE00) Mouse CXCL1/KC Quantikine ELISA Kit (Catalog # MKC00B) Sample: Mouse serum Application: ELISA

For the most up-to-date listing of products for Chemokine Family research | RnDSystems.com/Chemokine